多房棘球蚴RNA结合蛋白EWS的原核表达及其细胞定位与功能的初步鉴定  

Prokaryotic Expression of the RNA Binding Protein EWS in Echinococcus multilocularis and Preliminary Identification of Its Cellular Localization and Function

作　　者：严玉婷 郭小腊 孙玥 何学东 丁军涛[1] 郑亚东 YAN Yu-ting;GUO Xiao-la;SUN Yue;HE Xue-dong;DING Jun-tao;ZHENG Ya-dong(College of Life Science and Technology,Xinjiang University,Urumqi,Xinjiang,830046,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou,Gansu,730046,China;College of Animal Science and Technology and College of Veterinary Medicine,Zhejiang A&F University,Hangzhou,Zhejiang,311300,China)

机构地区：[1]新疆大学生命科学与技术学院,新疆乌鲁木齐830046 [2]中国农业科学院兰州兽医研究所,甘肃兰州730046 [3]浙江农林大学动物科技学院/动物医学院,浙江杭州311300

出　　处：《动物医学进展》2023年第12期29-35,共7页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31702224);浙江农林大学科研发展人才基金项目(2021LFR038)。

摘　　要：通过对多房棘球绦虫(Echinococcus multilocularis)RNA结合蛋白EWS(EmEWS)基因的原核表达、抗体制备、细胞定位等研究,初步探究EmEWS基因特征以及与RNA的结合能力。根据Wormbase数据库中EmEWS基因序列设计引物,利用RT-PCR扩增EmEWS基因并构建重组表达质粒,诱导表达、纯化重组蛋白,制备EmEWS多克隆抗体后进行Western blot、实时荧光定量PCR、免疫荧光定位、双荧光测定。结果表明,EmEWS基因长度为1485 bp,编码495个氨基酸,在多房棘球蚴原代细胞中表达量较高,在原头蚴中最低。生物信息学分析表明,细粒棘球蚴EWS与EmEWS相似性高达98.3%,且EmEWS的RBD结构域在绦虫中高度保守。EmEWS重组蛋白大小约为54 ku,制备的多克隆抗体可识别多房棘球绦虫中天然EmEWS蛋白。细胞定位显示EmEWS主要分布在细胞膜上。双荧光试验证明EmEWS可以与结合富含UG的RNA序列结合。综合上述结果,推测EmEWS可能作为转录调控因子参与小RNA的形成,同时有助于核蛋白的运输。Through prokaryotic expression,antibody preparation and tissue distribution of RNA binding protein EWS(EmEWS)gene of Echinococcus multilocularis(E.multilocularis),the gene characteristics and binding ability of EmEWS with RNA were preliminarily explored.A pair of primers were designed according to the sequence from WormBase.EmEWS gene was amplified by RT-PCR and a recombinant expression plasmid was constructed.The recombinant EmEWS protein was induced expressed and purified.EmEWS polyclonal antibody was prepared and identified by Western blot,real-time fluorescence quantitative PCR,immunofluorescence assay and dual fluorescence assay.The results showed that the full length of EmEWS gene was 1485 bp,encoding 495 amino acids.The EmEWS gene was highly expressed in the primary cells of E.multilocularis and lowest in the protoscoleces.Bioinformatics analysis showed that EWS of Echinococcus granulosus was 98.3%similar to EmEWS,and the RBD domain of EmEWS was highly conserved in tapeworms.The size of recombinant EmEWS protein was about 54 ku,and the prepared polyclonal antibody could specifically recognize the native EmEWS protein.Immunoflurescence showed that EmEWS mainly distributed on the cell membrane.Double fluorescence assay demonstrated that EmEWS could bind to UG-rich RNA sequences.Taken together,it is speculated that EmEWS may be involved in the formation of small RNA as a transcription regulator and contribute to nuclear protein transport.

关 键 词：多房棘球蚴 RNA结合蛋白EWS基因 原核表达 多克隆抗体 免疫荧光 

分 类 号：S852.724[农业科学—基础兽医学] Q789[农业科学—兽医学]